Using a number of complementary anatomical and biochemical techniques, the effects of hyperalgesia on the rat nervous system were studied. Lumbar spinal cord neurons were retrogradely labeled after injections of the inactive subunit of cholera toxin (subunit-B) into the caudal midbrain of normal rats. Spinal cord sections from these animals were processed for dynorphin (DYN) immunoreactivity. A larger percentage of projection neurons were contacted by DYN immunoreactive varicosities ipsilateral to a hyperalgesic hindpaw. This ipsilateral-contralateral difference was most pronounced in lamina I, where significantly more DYN immunoreactive varicosities were in apposition to retrogradely-labeled neurons. Spinal cord sections of animals with a hyperalgesic hindpaw were processed for glutamic acid decarboxylase (GAD) immunoreactivity. We observed that the number of neurons immunoreactive for GAD was substantially greater in spinal cord sections ipsilateral to the hyperalgesic hindpaw. In concurrence with this observation, an enzymatic-based spectrofluorometric assay of spinal cord revealed a 36% increase in gamma amino butyric acid (GABA) content ipsilateral to the hyperalgesic hindpaw. We examined alterations in the content of calcitonin gene-related peptide (CGRP) in primary afferents innervating a hyperalgesic hindpaw. It was found that there is significantly more CGRP within L4 dorsal root ganglia associated with the hyperalgesic vs. normal paw.